Today, insertion of predetermined points of attachment for an active moiety plays a key role for efficient binding of biological products. Conjugation of proteins and functional compounds is typically accomplished through covalent attachment of the functional compound to side chains of amino acid residues. Classical bioconjugate technologies are known to work in a non-site-restricted fashion which makes it difficult to avoid undesirable couplings at critical amino acid residues and also poses a risk for lot consistency due to inherent heterogeneity of the outcome of the coupling reaction. Non-selective chemical coupling via hydroxyl-groups of protein-linked carbohydrate structures requires harsh reaction conditions and bears the risk of unwanted modification of amino acid side chains of the protein. Critical residues that may be affected by undesirable coupling or modification include amino acid side chains important for overall product thermal stability and aggregation propensity. In case of antibodies, unwanted conjugation to amino acid side chains within or in proximity to the complementarity determining regions (CDRs) may lead to reduced affinity and heterogeneous antigen-binding properties. Chemical coupling is typically accomplished via protein-linked functional groups such as primary amines, sulfhydryls, carbonyls, carbohydrates, carboxylic acids and hydroxyl groups. Reactive groups used for coupling via such functional groups include aryl azide, carbodiimide, carbonyl, diazirine, hydrazide, hydroxymethyl phosphine, imidoester, isocyanate, maleimide, N-hydroxy-succinimide ester (NHS-ester), pentafluorophenyl ester (PFP-ester), psoralen and pyridyl disulfide.
Conjugation can be easily directed at sulfhydryl groups. However, due to the reactivity of the thiol group, free thiols are rarely found in expressed proteins. Direct labeling of free thiol-groups thus relies mainly on the reduction of existing disulfide (S—S) bonds. Antibodies as one of the major groups of therapeutic proteins have been coupled to functional compounds via sulfhydryl groups in the past. Since reduction of the heavy to light chain disulfide bond occurs at approximately double the frequency of the heavy to heavy disulfide bonds, such partial reduction approaches bear the possible risk of protein fragmentation by light chain loss. (Sun, et al., Bioconjug Chem 16:1282-1290 (2005).) In particular, the thermal stability of the critical antibody CH2 domain may be negatively affected by reduction of the inter-sheet disulfide bond. Production of a homogenous product from such random-type sulfhydryl-coupling reaction is a rather complicated and inefficient process. Early preclinical versions of the cAC 10 antibody drug conjugate, a sulfhydryl-linked immunoconjugate involved linkage of eight cytotoxic drug molecules per antibody molecule (Doronina et al., Nat. Biotechnol. 21(7): 778-84 (2003)). The coupling-enabled cysteine residues were obtained by reduction of the four interchain disulfide bonds (Doronina et al., Nat. Biotechnol. 21(7): 778-84 (2003)). Incomplete reduction of disulfide bonds led to a heterogeneous mixture of incomplete conjugates with less than eight drug molecules loaded per antibody (Hamblett et. al. Effects of drug loading on the antitumor activity of a monoclonal antibody-drug conjugate. Clinical Cancer Research, 2004, 10(20):7063-70). Product homogeneity for the random sulfhydryl-coupled conjugate proofed difficult to achieve and overall yield was rather low (Hamblett et. al. 2004).
Coupling via amino-groups of lysine residues is also a common mode of producing bioconjugates of proteins and functional compounds. Most recently, a thiol-containing maytansinoid, DM1 (N-methyl-N-[3-mercapto-1-oxopropyl]-L-alanine ester of maytansinol), an analogue of the clinically-studied drug maytansine) was used to link maytansinoids to antibodies through disulfide bonds (Barginear and Budman, 2009) In the case of Trastuzumab-DM1, DM1 is linked to trastuzumab using the bifunctional reagent, SMCC(N-succinimidyl-4-maleimidomethyl-cyclohexanecarboxylate.) SMCC is first added to lysine residues on the protein to produce a linker modified antibody. Coupling of the succinimidyl-group of SMCC to lysine residues happens in a random fashion targeted at all surface exposed lysine residues of the antibody. The thiol group in DM1 is then reacted with the maleimide group of the linker to form the nonreducible thioether bond (Barginear and Budman, 2009). Maleimides react with sulfhydryls at pH 6.5-7.5 to form stable thioether bonds. At pH values>7.5, however, maleimides also react toward primary amines which can result in the production of undesired covalent protein oligomers. In addition, the random coupling of SMCC to the antibody results in a non-homogenous bioconjugate product. The C-terminal lysine of antibody heavy chains is prone to clipping during upstream cell culture production and thus further coupling heterogeneitiy may result from differentially clipped C-terminal lysine residues. Each Trastuzumab-DM1 antibody contains an average of 3.5 drug molecules (Smith S V. Technology evaluation, Hun90′-dml, immunogen. Curr Opin Mol Ther 2005; 7: 394-401.), reflecting the typical distribution from 0 to 8 drug molecules per antibody (Blattler W A, Chari R V J, Vite G D, Altmann K H, Eds. Anticancer Agents—Frontiers in Cancer Chemotherapy, American Chemical Society, Washington 2001; 317-38.). The stoichiometric molar ratio of antibody and functional compound is an important determinator of therapeutic activity and conjugate stability. Kulkarni et al. (Cancer Research 41:2700-2706 (1981)) found that the highest efficient antibody-to-toxin-ratio obtained for methotrexate was about ten methotrexate molecules per antibody, and that attempts to increase the drug-antibody molar ratio beyond this threshold decreased the yield of immunoconjugate and damaged antibody activity. Similar results have been reported by Kanellos et al. (JNC 75:319-329 (1985)).
The inherent inhomogeneity of random-coupled bioconjugate products poses a challenge for stability studies, lot consistency and in-process analytics. Production of a homogenous bioconjugate product from a random coupling reaction can only be accomplished with significant downstream effort associated with a dramatic loss of product yield. Thus, there is a need for antibodies having one or more predetermined sites for stoichiometric attachment of functional compounds.
Recently, such antibodies for predetermined, site-directed thiol-coupling were described by Seattle Genetics Inc. (United States Patent Application 2008/0305044). While this mode of coupling has all the benefits of a site-directed approach, the minor destruction of tertiary structure is likely to impact overall product thermal stability.
United States Patent Application 2010/0254943 AMINO ACID SUBSTITUTED MOLECULES and related applications belonging to the same patent family disclose a method for obtaining site specific conjugates of proteins by incorporating coupling enabled non-natural amino acids into the protein sequence and for utilizing such non-natural amino acid residues as an anchoring position for further chemical or biological modification. The amino acid position at which the non-natural amino acid is incorporated is specified by a codon that is typically used to specify a naturally occurring amino acid (such as a wobble codon, a bias codon, a sixth box codon, a 4 box codon, or any other sense codon that the host cell or in vitro translation system might be used to specify a non-natural amino acid incorporation site), or a codon which is typically a stop codon, such as amber, ochre, or opal, or a frameshift codon. In cases where in-frame stop codons are used for artificial incorporation of non-natural amino acids, the cells need to be knocked-out for the cognate release factor (or translation termination factor). Given the redundancy between the existing translation termination factors, cells will always produce both the correct full length protein containing the incorporated non-natural amino acids and also prematurely truncated versions of the target protein. This makes this type of production method for such coupling enabled proteins highly inefficient, particularly in eukaryotic expression systems where the release factor eRF1 functions as an omnipotent release factor and recognizes all three termination codons. While this method is suitable to produce proteins enabled for site directed and defined coupling, there is still a need to produce such defined coupling-enabled proteins at far higher process efficiency.
Glycan-Structures linked to naturally occupied N-glycosylation sites typically stabilize protein conformation. The size of the attached glycan apparently has only a very minor impact on protein thermal stability (Shental-Bechor and Levy, 2009) which makes naturally occurring N-Glycans an ideal linker for the conjugation of functional compounds—even if such compounds have a high molecular mass. In line with this, U.S. Pat. No. 7,138,371 and United States Patent Application 2010/0048456 disclose methods for conjugating polypeptides via the protein linked glycostructure to polyethylene glycol. Both of these documents as well as related applications and patents still did not solve the problem that leads to coupling heterogeneity.
Thus, there is still a need for homogenous and stable protein-pharmaceutically active compound-conjugates, wherein the pharmaceutically active compounds are coupled via an exactly defined moiety to the proteins. Particularly, site-directed coupling of pharmaceutically active compounds to proteins via a predetermined sugar attachment site is desirable. There is also still a need for cells for producing proteins which comprise such an exactly defined coupling moiety in high yields and which, thus, allow homogeneous and efficient coupling of pharmaceutically active compounds in a high degree, methods for producing such proteins using said cells, and methods for producing conjugates comprising such proteins and pharmaceutically active compounds.
The inventors of the present invention surprisingly found that homogenous, efficient, stable and site-directed coupling of pharmaceutically active compounds to molecules such as lipids or proteins can be achieved via an artificial core fucose analogue introduced into the glycostructure of said molecules. They also surprisingly found that homogenous, efficient, stable and site-directed coupling of pharmaceutically active compounds to proteins, e.g. glycoproteins, can be achieved via an artificial fucose molecule linked to a protein-O-fucusylation site incorporated in or attached to the amino acid sequence of said proteins. They particularly provide cells and methods which allow the production of said molecules, e.g. proteins or lipids, and conjugates between said molecules, e.g. proteins or lipids, and a pharmaceutically active compound in high yields. The produced conjugates are thermally stable and homogenous. The molecule can be a glycoengineered protein, a therapeutic protein, an antibody, a vaccine component, even a protein comprised in the envelope of an enveloped virus. The advantageous unifying concept is that the molecule, e.g. protein or lipid, is produced by a cell that is engineered for impaired innate fucosylation to its proteome so that exogenously added fucose is preferentially incorporated at a specific site, and that this exogenously added fucose is chemically activated so that further pharmaceutically active compounds can be selectively and covalently attached to said molecule. The added pharmaceutically active compound can be a compound that enhances or transforms the properties of the molecule, e.g. protein such as an anitbody or lipid, for example, it can increase cytotoxicity, increase biological half life, induce targeting of the molecule to specific tissues, protect against degradation or aggregation, induce or enhance innate or adaptive immunity, or increase or decrease infectivity of live viruses.
In one or more aspects, embodiments, preferred embodiments or more preferred embodiments of the present invention described below, the particular fucose analogue comprising molecules, e.g. proteins or lipids, or the particular fucose analogue-linked conjugates with site-specific attachment of a molecule, e.g. protein or lipid, and a pharmaceutically active compound (e.g. immunoconjugates) may have one or more of the following advantages:    (I) A fucose analogue bound to the fucosylation site of the chitobiose core of proteins allows the coupling of a limited and defined number of pharmaceutically active compounds and, thus, enables the production of homogenously coupled conjugates. This positively influences product yield, lot consistency, therapeutic efficacy, and product comparability.    (II) The fucose analogue has only a very minor impact on overall protein thermal stability. Thus, therapeutic efficacy by avoiding inactive compounds can be increased.    (III) The artificial introduction of further defined N-glycosylation sites achieved due to the introduction of a single point mutation in the vicinity of a suitable asparagine residue, allows further site specific coupling of additional pharmaceutical compounds per protein molecule.    (IV) To link the pharmaceutically active compound via a fucose analogue which is bound to the fucosylation site of the chitobiose core is advantageous as the distal part of a glycan is accessible to enzymatic degradation and hence coupling at those distal sites would result in instability of the conjugate. Chemical homogeneity and coupling stability of the conjugate is achieved by direct coupling of a pharmaceutically active compound to a fucose analogue monosaccharide bound at a defined position in the polypeptide chain (C glycan or O-glycan) or in the glycan structure. In case of an N-linked glycan, the core fucose, i.e. the particular fucose residue alpha-1,6-linked to the reducing end of the first N-acetylglucosamine residue of the chitobiose core of an N-glycan constitutes the shortest possible, defined and homogenous glyco-linker for such an N-linked-glycoconjugate.    (V) The fucose analogue can be added to the culture medium. It is then taken up and further metabolized by conventional cells. However, the efficiency of the fucose de novo synthetic pathway starting from the abundant monosaccharide mannose (that itself is part of N glycans) provides 90% of the GDP fucose pool even in the presence of exogenous fucose and prevents efficient incorporation of fucose analogue into glycoproteins and inevitably results in a heterogeneous mixture of glycoproteins that are enabled for fucose-directed coupling to a low degree. Surprisingly, the inventors of the present invention found that cells with an interrupted biosynthesis pathway for fucose grown in a cell culture medium containing specific coupling-enabled fucose analogues efficiently incorporate the fucose analogue into glycoproteins produced in said cells. This results in a homogenous mixture of glycoproteins that are enabled for fucose-directed coupling to a high degree. In addition, the inventors found similar growth and performance parameters as the unmodified parental cell line grown in a medium not spiked with a coupling-enabled fucose analogue.    (VI) Unlike other coupling technologies that rely on incorporation of coupling-enabled artificial amino acids or sugars into a produced protein, the technology described herein does not suffer from the associated process yield decline typically seen for such modified proteins. In particular, the inventors did not observe a block of core-fucosylation as it was described in previous patent applications concerning fucose alkyne or azido-fucose. In contrast thereto, the inventors observed an unexpected and efficient incorporation of azido-fucose.    (VII) The covalent chemical bond between the protein bound fucose analogue and the conjugated pharmaceutically active compound is stable, not sensitive to mild reduction and therefore mitigates the risk of unwanted systemic release of the conjugated moiety.    (VIII) Apart from antibody drug conjugates that kill target cells via the attached toxin cell mediated cytotoxicity, antibody dependent cellular cytotoxicity (ADCC) is the dominating mechanism of action of therapeutic antibodies, e.g. IgG1-type therapeutic antibodies. Such antibody molecules should either allow coupling of a toxin or be equipped for efficient ADCC. It is further desirable that a drug conjugated molecule is disabled for ADCC to avoid toxicity directed towards effector cells. It is, thus, desirable for toxin-linked conjugates to combine both action principles in a single drug in particular if an antibody coupling efficiency does not reach 100%. The present invention may provide a solution. Linking the drug via a fucose analogue will obliterate effector functions such as ADCC and uncoupled antibodies within an antibody composition produced in the cells disclosed herein will lack fucose and therefore provides enhanced ADCC.    (IX) It is particularly advantageous if a fucose analogue which is directly linked to the polypeptide chain of a protein enables chemically homogenous coupling between said protein and a pharmaceutically active compound. This is achieved without modification of a protein that naturally contains a single or several protein-O-fucosylation sites. For proteins that do not contain protein-O-fucosylation sites or contain less sites than desired, the inventors of the present invention found the incorporation of an EGF-like repeat, representing such site, is very eligible.